Methods for treating subjects infected with a herpes virus or neisseria gonorrheae

ABSTRACT

The present invention provides a method of inhibiting the formation of infectious herpes virus particles, particularly infectious herpes simplex virus (HSV) particles, in a host cell. The method involves administering an effective amount of a hydroxylated tolan, particularly a polyhydroxylated tolan, to a herpes virus infected host cell. The present invention also provides a method of treating a herpes virus infection, particularly an HSV infection. The method comprises administering a topical composition comprising a therapeutically effective amount of a hydroxylated tolan to a herpes virus-infected site. The present invention also relates to a topical composition for treating a herpes virus infection selected from the group consisting of an HSV infection, a cytomegalovirus infection, and a varicella zoster virus infection. The present invention also provides a method of treating a subject infected with  Neisseria gonorrhea.

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/225,609, filed Aug. 15, 2000.

BACKGROUND

[0002] The present invention relates to compositions which inhibitreplication of herpes virus and the bacterium Neisseria gonorrheae, andmethods of using such compositions to treat subjects infected with thesemicroorganisms.

[0003] Human herpes viruses can infect host cells in virtually any organof the human body. Replication of a herpes virus within an infected hostcell leads to lysis of the infected cell and the release of largenumbers of infectious virus. The infectious particles released from thelysed cell can infect and destroy other cells at or near the site of theinitial infection. These infectious particles can also be transmitted toa non-infected individual. Human herpes viruses can also enter andremain latent, i.e., in the non-replicative state, in other cells of theafflicted individual for life. This life-long infection serves as areservoir of infectious virus for recurrent infections in the afflictedindividual and as a source of infection for an unwitting contact.

[0004] At least four of the human herpes viruses, including herpessimplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2),cytomegalovirus (CMV), and varicella zoster virus (VZV) are known toinfect and cause lesions in the eye of certain infected individuals.Together, these four viruses are the leading cause of infectiousblindness in the developed world.

[0005] HSV-1 primarily infects the oral cavity, while HSV-2 primarilyinfects genital sites. However, any area of the body, including the eye,skin and brain, can be infected with either type of HSV. Generally, HSVis transmitted to a non-infected individual by direct contact with theinfected site of the infected individual.

[0006] The initial symptoms of a primary or recurrent HSV infectioninclude tingling, pain, and/or parasthesia at the site of infection.This is followed by formation of a lesion at the infected site, i.e., inthe oral cavity, eye, skin, or reproductive tract. Healing typicallyoccurs in approximately ten to fourteen days.

[0007] The immune reaction that occurs in response to an HSV infectionprevents dissemination of the virus throughout the body of theimmmunocompetent individual. Such immune reaction, however, does noteliminate all infectious HSV particles from the body of the afflictedindividual. The virus particles that are not killed by the immuneresponse move along the nerve path to the ganglia of the infectedindividual where they remain in a state of latency. In response to avariety of stimuli including stress, environmental factors, othermedications, food additives or food substances, the infectious virusparticles may leave the ganglia and cause a recurrent infection at ornear the original site of infection. In those HSV-infected individualswho are immunosuppressed or who lack a well-developed immune system,such as neonates, dissemination of the virus particles from the infectedsite can also occur and lead to life-threatening complications,including encephalitis.

[0008] VZV, which is transmitted by the respiratory route, is the causeof chickenpox, a disease which is characterized by a maculopapular rashon the skin of the infected individual. As the clinical infectionresolves, the virus enters a state of latency in the ganglia, only toreoccur in some individuals as herpes zoster or “shingles”. Thereoccurring skin lesions remain closely associated with the dermatome,causing intense pain and itching in the afflicted individual.

[0009] CMV is more ubiquitous and may be transmitted in bodily fluids.The exact site of latency of CMV has not been precisely identified, butis thought to be leukocytes of the infected host. Although CMV does notcause vesicular lesions, it does cause a rash.

[0010] There are no known cures for infections with human herpesviruses, i.e., methods of eliminating the virus from the body of theinfected individual. In addition, there are very few methods forblocking the formation of infectious herpes virus particles and therebyreducing the frequency, severity, or duration of a herpes virus-inducedinfection and the likelihood of recurrence of infection in thelatently-infected individual. Thus, it is desirable to have additionalmethods for inhibiting the formation of infectious herpes virusparticles. Such method is useful for limiting the severity of a herpesvirus infection within an infected individual and the likelihood oftransmission of the herpes virus infection from the infected individualto a non-infected individual.

[0011]Neisseria gonorrhea is a gram negative bacterium that ispathogenic in humans. The bacterium is spread from person to person bycontact with infected secretions, most often by sexual contact. Once thepathogen is deposited on a mucosal surface, a complex series ofmolecular interactions occur that result in invasion of mucosal columnarcells. The spectrum of diseases ranges from local infections of theurethral, cervical, rectal and oropharyngeal membranes to invasion ofthe pelvis or epididymis, to invasion of the blood stream, with orwithout dissemination to distant organs such as heart valves, joints,and pericardium. The pathogen may also infect the conjunctiva.Gonococcal conjunctivitis is most often contracted by neonates passingthrough an infected birth canal, although adults can also be infected.

[0012] The quest for a gonococcal vaccine has been ongoing for manyyears with virtually no success. Accordingly, the primary treatmentinvolves preexposure or postexposure antibiotic prophylaxis. In additionto antibiotic eyedrops, silver nitrate has also been used to treatneonatal gonococcal conjunctivitis. Unfortunately, the bacterium hasdeveloped resistance to some of the most common antibiotics, such aspenicillin. Accordingly, additional compositions for reducing growth ofthis pathogen are desirable.

SUMMARY OF THE INVENTION

[0013] The present invention provides a new method of inhibiting theformation of infectious herpes virus particles, particularly infectiousHSV particles, in a host cell. The method involves administering ahydroxylated tolan, particularly dihydroxytolan or trihydroxytolan, to aherpes virus infected host cell. The hydroxylated tolan is administeredto the host cell in an amount sufficient to inhibit replication of thevirus in the virus-infected host cell. Such method is useful forreducing the cytopathic effect of a herpes virus infection. Such methodis also useful for preventing the spread of the herpes virus from avirus-infected host cell to a non-infected host cell. Such method isalso useful for establishing a model system for studying the molecularevents that occur during replication of herpes virus and for studyingthe factors that trigger replication of a latent herpes virus,particularly replication of latent HSV.

[0014] The present invention also provides a method of treating asubject having or suspected of having a herpes virus infection,particularly an HSV infection. The method comprises administering atopical composition comprising a therapeutically effective amount of ahydroxylated tolan, particularly a di-hydroxylated or tri-hydroxylatedtolan, to a herpes virus-infected site. The present invention alsorelates to a topical composition for treating a herpes virus infectionselected from the group consisting of an HSV infection, a CMV infection,and a VZV infection.

[0015] The present invention also provides a method of inhibitingreplication of the gram negative bacterium Neisseria gonorrhea. Suchmethod involves contacting the bacterium with a composition containing ahydroxylated tolan, preferably a di-hydroxylated or tri-hydroxylatedtolan. In vivo, such method can be used to treat an individual who hascome in contact with, (e.g., a carrier), or an individual who isexpected to come into contact with the bacterium. In vivo, such methodcomprises administering a composition comprising a therapeuticallyeffective amount of a hydroxylated tolan, particularly atri-hydroxylated tolan to the subject

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 shows the structures of 4,4′ dihydroxytolan (“Tolan-5”),3,4′,5-trihydroxytolan (“Tolan-10”) and4-hydroxy-4′-trifluoromethyltolan (“Tolan-11).

[0017]FIG. 2 is a synthetic scheme for making hydroxylated tolans.

[0018]FIG. 3 is a synthetic scheme for making hydroxylatedtrifluoromethyltolans.

[0019]FIG. 4 is a graph showing the extent of HSV-1 replication invirus-infected cells treated with different concentrations of adihydroxylated tolan.

[0020]FIG. 5 is a graph showing the extent of HSV-1 replication invirus-infected cells treated with 4,4′ dihydroxytolan at different timesfollowing infection.

[0021]FIG. 6 is a graph depicting the reversibility of the inhibitoryeffect of 4,4′-dihydroxytolan on HSV-1 replication in virus-infectedcells.

[0022]FIG. 7 is a graph depicting the effect of treatment with4,4′-dihydroxytolan on the accumulation of ICP-4 in HSV-1 infectedcells.

[0023]FIG. 8 is a graph depicting the effect of treatment with4,4′-dihydroxytolan on the accumulation of ICP-27 in HSV-1 infectedcells.

[0024]FIGS. 9A, 9B, and 9C are graphs showing the inhibitory effects of4,4′ dihydroxytolan (“Tolan-5”), 3,4′,5-trihydroxytolan (“Tolan-10”) and4-hydroxy-4′-trifluoromethyltolan (Tolan-11), respectively, onreplication of HSV-1 in infected cells.

[0025]FIG. 10 is a graph showing the effect of a 5% Tolan-10 solution onthe development of herpetic lesions in animals infected with HSV-1.

[0026]FIG. 11 is a graph showing the effect of a 10% Tolan-10 solutionon the development of herpetic lesions in animals infected with HSV-1.

[0027]FIG. 12 is a graph showing the percentage of control animals andtolan-treated animals that survive an infection with HSV-1.

DETAILED DESCRIPTION OF THE INVENTION

[0028] In one aspect, the present invention provides a method ofinhibiting formation of infectious herpes virus particles, particularlyinfectious HSV particles, in a host cell. The method comprisesadministering a hydroxylated tolan to the host cell. The hydoxylatedtolan is administered in an amount sufficient to or effective to inhibitreplication of the herpes virus within the infected cell. Preferably,the hydroxylated tolan, is administered to the host cell either prior toinfection of the host cell with the virus or within six hours afterinfection of the host cell with the virus.

[0029] Preferably, the hydroxylated tolan is administered to the hostcell by contacting the host cell with or exposing the host cell to acomposition comprising the hydroxylated tolan. For example, in vitro,the method comprises adding a hydroxylated tolan to the culture mediumof herpes virus-infected host cells. In the case of cultured cells, thehydroxylated tolan preferably is added to the medium before the hostcells are infected with the virus or within six hours after the hostcells are infected with the virus. In the case of ganglia, which servesas an organ culture model system for studying latency of herpes viruses,particularly for studying latency of HSV, the hydroxylated tolan isadded to the medium after the ganglia are excised from thelatently-infected host.

[0030] It has been determined that treatment of cultured cells inaccordance with the present method is non-toxic to cells and blocksreplication of HSV at some early stage in the replicative cycle of thishuman herpes virus. It has also been determined that the effect of apolyhydroxylated tolan on HSV replication is reversible. Typical of theherpes viruses, HSV replication occurs in phases, with each phase beingdependent on the successful completion of the prior phase. The“immediate early phase” occurs at 1-3 hours after infection and isassociated with regulatory and synthetic events. The “early phase”occurs 3-6 hours after infection and is also associated with regulatoryand synthetic events, particularly the synthesis of virus DNA. The “latephase” occurs 6-10 hours after infection and is associated with finalsynthetic events and assembly of viral components into infectionsvirions. Accordingly, since all herpes viruses have in common areplicative scheme that progresses through similar and distinct phases,such method is useful for establishing model systems for studying themolecular events that occur during replication of all herpes viruses.For example, mammalian cell cultures incubated in the presence andabsence of a hydroxylated tolan may be used to identify cellular factorsthat are involved in regulating herpes virus synthetic events. Such cellcultures may also be employed to characterize the role of HSV geneproducts in the replication of infectious virus, particularly thoseproteins and factors whose function are currently unknown.

[0031] Such method is also useful for establishing a model system forstudying latency of herpes viruses, particularly latency of the herpesviruses that remain latent in the ganglia, such as for example HSV andVZV. Such model system is useful for characterizing the extracellularfactors such as for example hormones and cytokines, as well as theintracellular factors and molecular events that trigger replication oflatent herpes viruses.

[0032] Methods and Compositions for Treating a Subject With aHerpesvirus Infection

[0033] In another aspect, the present invention provides methods fortreating a subject with a herpesvirus infection. The method comprisesadministering a pharmaceutical composition, preferably a topicalcomposition, comprising a therapeutically effective amount of ahydroxylated tolan, preferably a dihydroxylated tolan or trihydroxylatedtolan, to the site of the infection. As used herein “site of theinfection” means a previously uninfected site which is expected to comeinto contact with a herpes virus-infected site or the site of a currentor prior herpes virus-induced lesion. Such method is particularly usefulfor treating local herpes virus infections, such as for example,HSV-induced skin lesions, HSV-induced eye infections, HSV-inducedlesions of the reproductive tract, CMV-induced eye lesions, andVZV-induced eye lesions. In such cases, it is preferred that thehydroxylated tolan be applied directly to the infected site. It ispreferred that the hydroxylated tolan be administered to theherpesvirus-infected site in the form of an aqueous solution or in theform of a salve. For eye infections, it is preferred that an aqueoussolution of the hydroxylated tolan, be administered as an eye drop. Forherpesvirus skin lesions, such as for example, HSV-induced skin lesions,or HSV-induced lesions of the reproductive tract, it is preferred thatthe composition be applied topically.

[0034] Method of Inhibiting Growth of Neisseria gonorrheae

[0035] In another aspect, the present invention provides a method ofinhibiting the growth of Neisseria gonnorhea. The method comprisesadministering a hydroxylated tolan, preferably a dihydroxlated ortrihydroxylated tolan to a surface which has come in contact with orcould come in contact with the organism. In vivo, the method, whichcomprises administering the hydroxylated tolan to a mucous membrane of ahuman subject, may be used to prevent or reduce the symptoms ofgonococcal disease in the human subject. The hydroxylated tolan may beincorporated into a pharmaceutical composition which is applied to themucous membrane of a carrier of the bacterium or a person who could comeinto contact with the carrier.

[0036] Administration of the pharmaceutical composition to an uninfectedsubject is via local administration to a site which has been or may becontacted with the pathogenic organism. It is preferred that thepharmaceutical composition be applied prior to exposure to the targetedpathogen or preferably within 1-24 hours, more preferably within 1-12hours after exposure of the uninfected subject to the pathogenicorganism. Administration of the pharmaceutical composition to a carrierof Neisseria gonorrhea is via local administration to the genitalia,rectum, or oropharynx.

Hydroxylated Tolans

[0037] The structural skeleton of the compound employed in the presentinvention, i.e., the hydroxylated tolan, comprises two aromatic ringsjoined by an acetylene bridge. Preferably, the hydroxylated tolan is apolyhydroxylated tolan, more preferably a dihydroxytolan, or atrihydroxytolan, most preferably a trihydroxytolan. A general scheme forpreparing polyhydroxylated tolans is shown in FIG. 2. A general schemefor preparing a hydroxylated trifluormethyltolan is shown in FIG. 3.

Topical Composition

[0038] The pharmaceutical composition comprises a therapeuticallyeffective amount of a hydroxylated tolan, preferably a polyhydroxylatedtolan, more preferably a dihydroxylated or trihyroxylated tolan, and apharmaceutically acceptable carrier, preferably a topical carrier.Preferably, the composition comprises a relatively inert topicalcarrier. Many such carriers are routinely used and can be identified byreference to pharmaceutical texts. Examples include polyethyleneglycols, polypropylene copolymers, and some water soluble gels. Such acomposition, referred to hereinafter as the “topical composition”, mayalso contain diluents, fillers, salts, buffers, stabilizers,solubilizers, and other pharmaceutically acceptable materials well knownin the art. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of the antiviralor antibacterial activity of the hydroxylated tolan.

[0039] In practicing the present method of treatment or use, apharmaceutical composition comprising a therapeutically effective amountof the hydroxylated tolan is applied to the site of infection in thehost subject before or after the host subject is exposed to the virus orbacterium. Such composition is particularly effective in treatinginfections of the eye, oral cavity and vagina as well as border areas ofthe lips and rectum. In the case of oral administration, dentrifices,mouthwashes, tooth paste or gels, or mouth sprays are used. Vaginal orrectal administration may be by the usual carriers such as douches,foams, creams, ointments, jellies, and suppositories, the longer lastingforms being preferred. Ocular administration is preferably by ophthalmicointments or solutions. Lip treatment is, preferably, in the form of agel.

[0040] The topical composition may further contain other agents whicheither enhance the activity of the hydroxylated tolan or complement itsactivity or use in treating the viral disease or bacterial disease. Suchadditional factors and/or agents may be included in the pharmaceuticalcomposition to produce a synergistic effect with the hydroxylated tolan,or to minimize side effects. The topical composition may also contain anagent which enhances uptake of the hydroxylated tolan.

[0041] Preferably the topical composition comprises a solvent for thehydroxylated tolan, such as, for example, an alcohol. A liquid carriersuch as water, petroleum, oils of animal or plant origin such as peanutoil, mineral oil, soybean oil, or sesame oil, corn oil, or syntheticoils may be added. The liquid form of the pharmaceutical composition mayfurther contain a physiological saline solution, dextrose or othersaccharide solution, or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol. The preparation of such topicalcomposition having suitable pH, isotonicity, and stability, is withinthe skill in the art.

[0042] The topical composition of the invention may be in the form of aliposome in which the hydroxylated tolan is combined with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art.

Dosage

[0043] The hydroxylated tolan is administered to the site of infectionin the host subject in a therapeutically effective amount. As usedherein, the term “therapeutically effective amount” means the totalamount of the hydroxylated tolan that is sufficient to show a meaningfulbenefit, i.e., treatment, healing, prevention, amelioration, orreduction in the symptoms of the herpesvirus or gonococcal infection oran increase in rate of healing, amelioration or reduction in thesymptoms of such infection.

[0044] By “treating” is meant curing or ameliorating a herpesvirus orgonococcal infection or tempering the severity of the infection. Bypreventing is meant blocking the formation of a primary lesion orrecurrence of a lesion at the infected site. The dosages of thehydroxylated tolan, which can treat or prevent an HSV, VZV, CMVinfection, or gonococcal infection, can be determined in view of thisdisclosure by one of ordinary skill in the art by running routine trialswith appropriate controls. Comparison of the appropriate treatmentgroups to the controls will indicate whether a particular dosage iseffective in preventing or treating the infection at the levels used ina controlled challenge.

[0045] It is contemplated that the various pharmaceutical compositionsused to practice the method of the present invention should containabout 0.1 μg to about 100 mg/ml. Although a single application of thetopical composition may be sufficient to ameliorate the pathologicaleffects of the herpesvirus or Nisseria gonorrhea, it is expected thatmultiple doses will be preferred.

Delivery

[0046] Administration of the pharmaceutical composition is via localadministration to the infected site. In those individuals who haveexperienced a primary lesion, it is preferred that the topicalcomposition be applied at the prodromal stage of infection, i.e., duringearly symptoms of pain, tingling, parasthesia. Preferably, thecomposition is applied to the site of infection periodically, morepreferably every three hours. The duration of therapy using thepharmaceutical composition of the present invention will vary, dependingon the severity of the disease being treated and the condition andresponse of each individual patient. Ultimately the attending physicianwill decide on the appropriate duration of using the pharmaceuticalcomposition of the present invention.

[0047] The following examples are for purposes of illustration only andare not intended to limit the scope of the claims which are appendedhereto. All references cited herein are specifically incorporated intheir entirety herein.

EXAMPLE 1 Synthesis of Poly-Hydroxylated Tolans

[0048] A. Synthesis of 3,5-dimethoxyiodobenzene from3,5-dimethoxyaniline

[0049] In a 500 ml 3-necked, round-bottomed flask equipped with athermometer, a mechanical stirrer and an addition funnel was placed HCl(12 M, 100 ml, 1.2 mol) and crushed ice (100 g). The flask was immersedin a dry ice-Me₂CO cooling bath, and 3,5-dimethoxyaniline (15.3 g, 100mmol) was added with stirring. To this cold mixture NaNO₂ (8.4 g, 120mmol) in 40 ml H₂O was added dropwise at such a rate to maintain thetemperature of the reaction mixture between −10˜−5° C. throughout theaddition. The reaction mixture was stirred for 1 hour at 0˜5° C. The reddark solution of the diazonium salt was added to a well-stirred solutionof KI (83 g, 500 mmol) in 200 ml H₂O at room temperature. The mixturewas stirred for 2 hours, then allowed to stand overnight. The resultingsolution was extracted with ether (200 ml×4). The pooled organicextracts were washed with brine (200 ml×2), and an aqueous saturatedNa₂S₂O₃ solution (200 ml×2), dried over MgSO4, filtered and concentratedto a small volume. Silica gel was added, and the mixture evaporated todryness. This preloaded silica gel was placed on a pad of silica gel andeluted with petroleum to give 17.5 g (66%) of a colorless solid,3,5-dimethoxyiodo benzene. ¹HNMR (CDCl₃, 300 Mz): δ ppm: 6.85 (2H, d,J=2.3, Ar—H), 6.40 (1H, t, J=2.3 Ar—H), 3.76 (s, 6H, 2CH₃O).

[0050] B. Synthesis of Arylethynyltrimethylsilanes fromEthylnyltrimethylsilane and Aryl Iodides

[0051] General Procedure

[0052] To a solution of aryl methoxy substituted aryl iodide (40 mmol)in isopropylamine (250 ml) were added Pd(PPh₃)₂Cl₂(0.4 mmol) and CuI(0.8 mmol), then trimethylsilylacetylene (44 mmol). The reaction mixturewas stirred at ambient temperature for 2-4 hours under a slow stream ofnitrogen. The reaction mixture was filtered and the residues were washedwith ethyl acetate, and the solvent evaporated from the combinedfiltrates. The crude product was purified by column chromatography onsilica gel using petroleum/ethyl acetate as an eluent to give themethoxy substituted arylethylyl trimethylsilanes.

[0053] (1) 2-(4-methoxyphenyl)-1-trimethylsilyl-ethyne

[0054] Purified by column chromatography on silica gel using petroleumether as an eluent to give 2-(4-methoxyphenyl)-1-trimethylsilyl-ethyne(96% yield) as a light yellow oil.

[0055] (2) 2-(3,5-dimethoxyphenyl)-1-trimethylsilyl-ethyne

[0056] Purified by column chromatography on silica gel using petroleumether as an eluent to give 2.2 g (94%) light yellow needles.

[0057] T_(GC)=5.39 (T_(init)=50° C.). ¹HNMR(CDCl₃, 300 Mz) δ ppm: 6.6(s,2H, Ar—H), 6.43(s, 1H, Ar—H), 3.77(s, 6H, 2CH₃), 0.24(s,9H, SiMe₃).

[0058] C. Synthesis of Methoxy Substituted Arylacetylenes

[0059] To a solution of arylethynyltrimethylsilanes (30 mmol) inmethanol (30 ml) was added potassium fluoride (3.5 g, 60 mmol). Thereaction mixture was stirred at room temperature for 2 hours. Afterremoval of methanol, the product was extracted with ether (100 ml×3) andpurified by chromatography on silica gel using petroleum ether as eluentto afford pure products.

[0060] (1) p-Methoxyrthylnylbenzene

[0061] Pale yellow oil was obtained in 92% yield.

[0062]¹HNMR (CDCl₃, 300 Mz): δ ppm: 7.94(d, 2H, J=8.98, Ar—H), 6.83(d,2H, J=8.55, Ar—H), 3.80(s, 3H, Ch₃O), 3.00 (s, 1H—H).

[0063] (2) 3,5-Methoxyrthylnylbenzene

[0064] Pale yellow needle was obtained in 91 % yield.

[0065]¹HNMR (CDCl₃, 300 Mz): δ ppm: 7.94(d, 2H, J=2.4, Ar—H), 6.83(d,2H, J=2.3, Ar—H), 3.78(s, 6H, 2Ch₃O), 3.94 (s, 1H—H).

[0066] D. Synthesis of Methoxytolans

[0067] General Procedure

[0068] To a solution of methoxyethylnylbenzenes (20 mmol) and methoxysubstituted aryl iodide (22 mmol) in isopropylamine (120 ml) were addedPd(PPH₃)₂Cl₂ (0.2 mmol) and CuI (0.4 mmol). The reaction mixture wasstirred at ambient temperature for 6 hours under a slow stream ofnitrogen. The reaction mixture was filtered and the residues were washedwith ethyl acetate and the solvent evaporated from the combinedfiltrates. The crude product was purified by column chromatography onsilica gel using petroleum ether/ethyl acetate (9:1) as an eluent togive methoxytolans.

[0069] (1) 3,4′,5-Trimethoxyltolan

[0070] A pale yellow oil was obtained in 93% yield.

[0071]¹HNMR (CDCl₃) 300 Mz): δ ppm: 7.46(d, 2H, J=8.6, Ar—H), 6.88(d,2H, J=8.8, A—H), d, 2H J=2.3, Ar—H), 6.44(t, 2H, J=2.3, Ar—H), 3.83(s,3H, CH₃O), 3.80(s, 6H, 2CH₃O).

[0072] (2) 3,3′,5,5′-Tetramethoxytolan

[0073] A colorless needle crystal was obtained in 85% yield.

[0074]¹HNMR (CDCl₃) 300 Mz): δ ppm: 6.69(d, 4H, J=2.3, Ar—H), 6.46(d,2H, J=2.3, Ar—H), 6.66(d, 2H J=2.3, Ar—H), 3.80(s, 12H, 4CH₃O).

[0075] (3) 4,4′-Dimethoxytolan

[0076] A colorless needle crystal was obtained in 91% yield.

[0077]¹HNMR (CDCl₃) 300 Mz): δ ppm: 7.46(d, 4H, J=8.7, Ar—H), 6.87(d,2H, J=8.7, Ar—H), 3.82(s, 6H, 2CH₃O).

[0078] E. Synthesis of Hydroxytolans

[0079] General Procedure

[0080] In a dry 250 ml, 3-necked, round-bottomed flask was placed asolution of methoxytolans (10 mmol) in anhydrous methylene chlorideunder N₂. The reaction mixture was cooled to below −20° C., and BBr₃ (20mmol×the number of methoxy groups) by syringe. Then the reaction mixturewas permitted to warm up to room temperature and stirred for over 24hours. The reaction mixture (a reddish clear solution) was then pouredinto ice-water and stirred. After sufficient stirring, an aqueous NaHCO₃solution was added to adjust the pH of the mixture to 7-8. Then themixture was extracted with ethyl acetate 3-4 times. The organic layerwas washed with brine and dried over MgSO₄. Solvent was removed underreduced pressure. The red brown color crude products was purified bycolumn chromatography on silica gel using petroleum/ethyl acetate (1:1)as en eluent to give hydroxytolans.

[0081] (1) 3,4′,5-Trihydroxytolan

[0082] A pale yellow solid was obtained in 82% yield.

[0083]¹HNMR (CDCl₃) 300 Mz): δ ppm: 9.89(s, 1H, OH), 9.45(s, 2 h, 2-OH,7.33(d, 2H, J=8.65, Ar—H), 6.78(d, 2H, J=8.63, Ar—H), 6.31(d, 2H, J=2.2,Ar—H), 6.23(d, 2H, J=2.2, Ar—H).

[0084] (2) 3,3 ,5,5′-Tetrahydroxytolan

[0085] A pale red solid was obtained in 92% yield.

[0086]¹HNMR (CDCl₃, 300 Mz): δ ppm: 9.49(s, 4H, 4-OH), 6.33(d, 4H,J=2.2, Ar—H), 6.25(t, 2H, J=2.2, Ar—H).

[0087] (3) 4,4′-Dihydroxytolan

[0088] A white solid was obtained in 93% yield.

[0089]¹HNMR (CDCl₃, 300 Mz): δ ppm: 9.82(s, 2H, 2-OH), 7.31(d, 4H,J=8.7, Ar—H), 6.77(d, 4H, J=8.7, Ar—H).

[0090] References

[0091] 1. Ali, M. A., Kondo, K. and Tsuda, Y., Chem. Pharm. Bull., 1992,40, 1130-1136.

[0092] 2. Pavia, M. R., et. al., Bioorg. Med. Chem., 1996; 4, 659-666.

[0093] 3. Jeffery, T., Tetrahedron Lett., 1994, 35, 3051-3054.

[0094] 4. Jeffery, T. and Galland, J. C. Tetrahedron Lett., 1994, 35,4103-4106.

[0095] 5. Schmidt-Radde, R. H. and Vollhardt, K. P. C., J. Am. Chem.Soc., 1992, 114, 9713-9715.

[0096] 6. Schumm, J. S., Pearson, D. L. and Tour, J. M., Angew. Chem.,Int. Ed. Engl., 1994, 33, 1360-1363.

[0097] 7. Pal, M. and Kundu, N. G., J. Chem. Soc., Perkin Trans., 1996,1, 449-451.

[0098] 8. Bumagin, N. A., Sukhomlinova, L. I., Luzikova, E. V., TolstayaT. P. and Beletskaya, I. P., Russ. J. Org. Chem., 1996, 32, 996-1000.

[0099] 9. Bumagin, N. A., Sukhomlinova, L. I., Luzikova, E. V., TolstayaT. P. and Beletskaya, I. P., Tetrahedron Lett., 1996, 37, 897-900.

[0100] 10. Meier H. and Dullweber, U., J. Org. Chem., 1997, 62,4821-4826.

EXAMPLE 2 Synthesis of 4-Hydroxy-4′-trifluoromethyltolan

[0101]FIG. 3 shows a synthetic scheme for the preparation ofhydroxy-trifluoromethyltolan. Synthetic details of the specific reactionsteps are described below. Most of the reactions were accomplished withhigh yields (over 90%). All products were purified by columnchromatography and characterized by GC and ¹HNMR spectrometry.

[0102] 1. 1-Iodo-4-tetrahydropyranyloxybenzene 1

[0103] To a stirred solution of 4-iodophenol (11.0 g, 50 mmol) in CH₂Cl₂(50 ml) cooled with an ice bath, dihydropyran (5.0 g, 60 mmol) was addeddropwise over 10 min at 0-5° C. After the solution became clear,toluenesulfonic acid, TsOH, (10 mg) was added. The solution was stirredat 20° C. for 15 min. Then it was quenched by addition of NaHCO₃ (1 g)and 3 drops of water, and after stirring for 5 min at 20° C., thesolvent was removed in vacuo and the residue was purified by columnchromatography on silica gel with petroleum ether as eluent to give 14.0g (92%) of 1 as colorless crystal; mp 66° C.; δ_(H)(CDCl₃; 300 MHz):7.55(d, J=8.3, 2H, Ar—H), 6.83(d, J=8.4, 2H, Ar—H), 5.37(t, J=3.1, 1H,OCHO), 3.86(m, 1H, THP), 3.59(m, 1H, THP), 1.87˜1.58(m, 6H, THP).

[0104] 2. 4-Tetrahydropyranyloxy-1-(trimethylsilylethynyl)benzene 2

[0105] To a degassed solution of compound 1 (9.12 g, 30 mmol) indiisopropylamine (180 ml) under nitrogen, Pd(PPh₃)₂Cl₂ (140 mg, 0.2mmol) and CuI (78 mg, 0.4 mmol) were added. Then trimethylsilylacetylene (3.3 g, 33 mmol) was added dropwise to this clear solution.The reaction mixture was stirred for 2 hours at room temperature. Thesalt formed during the reaction procedure was filtered off and washedwell with ethyl acetate. The filtrate was evaporated to dryness andhydrolyzed with concentrated hydrochloric acid (5 ml), water (25 ml) andcrushed ice (10 g), then extracted with ethyl acetate. The combinedorganic paste was washed with brine and dried with MgSO₄. The solventwas removed in vacuo and the residue was purified by columnchromatography (petroleum ether-ethyl acetate=9:1) to give a yellow oilof 2. Yield 7.9 g (96%); δ_(H)(CDCl₃; 300 MHz): 7.39(d, J=8.7, 2H, Ar—H,6.97(d, J=8.6, 2H, Ar—H), 5.41(t, J=3.1, 1H, OCHO), 3.84(m, 1H, THP),3.59(m, 1H, THP), 1.86˜1.61(m, 6H, THP), 0.23(s, 9H, 3CH3).

[0106] 3. 4-Tetrahydropyranyloxyphenylacetylene 3

[0107] KF (9.3 g, 160 mmol) was added to a stirred solution of 2 (22.6g, 80 mmol) in MeOH (150 ml). The reaction mixture was stirred at roomtemperature for about 4 hours. After the reaction finshed (GC shows nostarting material remaining), the solvent was removed under reducedpressure on a rotary evaporator. The residue was purified by columnchromatography on silica gel (petroleum ether-ethyl acetate=9:1) to givea pale yellow crystals of 3. Yield 15.7 g(97%); mp 65° C., δ_(H)(CDCl₃;300 MHz): 7.42(d, J=8.7, 2H, Ar—H), 7.00(d, J=8.7, 2H, Ar—H), 5.43(t,J=3.2, 1H, OCHO), 3.87(m, 1H, THP, 3.60(m, 1H, THP), 2.99(s, 1H, C═C—H),1.96˜1.56(m, 6H, THP).

[0108] 4. 4-Tetrahydropyranyloxy-4′-trifluormethyltolan 4

[0109] A solution of 3 (12.1 g, 60 mmol) and 4-bromobenzotriflouride(14.85 g, 66 mmol) in diisopropylamine (250 ml) was heated to 30° C.under nitrogen, and the solution was degassed. Then Pd(PPh₃)₂Cl₂ (210mg, 0.3 mmol) and copper(I) iodide (114 mg, 0.6 mmol) were added to thisclear solution. The reaction mixture was stirred for 2 hours at 80° C.,then cooled to room temperature. The salt formed during the reactionprocedure was filtered off and washed well with ethyl acetate. Thefiltrate was evaporated to dryness and hydrolyzed with concentratedhydrochloric acid (10 ml), water (100 ml) and crushed ice (50 g), thenextracted with ethyl acetate. The combined organic paste was washed withbrine and dried over MgSO₄. The solvent was removed in vacuo and theresidue was purified by column chromatography (petroleum ether-ethylacetate=9:1) to give a pale yellow crystals of 4. Yield 16.6 g (80%); mp112˜113° C.; δ_(H)CDCl₃; 300 MHz): 7.59(s, 4H, Ar—H), 7.48(d, J=8.7, 2H,Ar—H), 7.04(d, J=8.7, 2H, Ar—H), 5.46(t, J=3.1, 1H, OCHO), 3.89(m, 1H,THP), 3.62(m, 1H, THP), 1.86˜1.62(m, 6H, THP).

[0110] 5. 5-Hydroxy-4′-trifluoromethyltolan 5

[0111] Compound 4 (13.84 g, 40 mmol), CH₂Cl₂ (75 ml) and MeOH (125 ml)were placed in a 250 ml round bottomed flask, then TsOH (0.4 g, 0.4mmol) was added. The reaction mixture was stirred at 30° C. for 1 hour.When the reaction was finished (TLC shows no starting materialremaining), the solvent was removed by rotary evaporation and theresidue was dissolved in EtOAc and filtered through silica gel. Thesolvent was removed and the solid was recrystallized from solvents ofethyl acetate and hexane (1:5) to give a pale yellow crystal 9.5 g(90%), mp 131-132° C., δ_(H)(CDCl₃; 300 MHz): 7.59(s, 4H, Ar—H), 7.44(d,J=8.7, 2H, Ar—H), 6.82(d, J=8.7, 2H, Ar—H), 5.16(s, 1H, OH).

[0112] References

[0113] 1 Shen, D., Diele, S., Pelzl, G., Wirth, I. and Tschierske, C.,J. Matter. Chem., 1999, 9, 661.

[0114] 2 Praefcke, K., Kohne, B. and Singer, D., Angew. Chem. Int. Ed.Engl., 1990, 29, 177.

[0115] 3 Bouchta, A., Nguyen, H. t., Achard, M. F., et al., Liq.Crystals, 1992, 12, 575.

[0116] 4 Hsieh, C. J. and Hsiue, G. H., Liq. Crystals, 1994, 16, 469.

EXAMPLE 3 Inhibiting Formation of Infectious HSV-1 Particles byTreatment With a Di-Hydroxylated Tolan

[0117] Cultures of African green monkey kidney cells (Vero) cells,obtained from the American Type Culture Collection, Rockville, Md., weregrown to confluence in Medium 199 supplemented with 5% fetal bovineserum, 0.075% NaHCO₃, and 50 μg/ml gentamycin sulfate in 25 cm² tissueculture flasks. Cells were infected with HSV-1 at a multiplicity ofinfection (moi) of one and incubated at room temperature for one hour toallow for virus attachment to and penetration of the cell. Under theseconditions, approximately half of the cells are infected with virus.Thereafter, the cultures were rinsed three time with media and incubatedin medium containing 50 μM or 75 μM 4,4′-dihydroxytolan prepared in 0.2%dimethyl-sulfoxide (DMSO). Controls were treated identically, but wereincubated without the dihyroxylated tolan. For purposes of comparison,HSV cell were also inoculated in the presence of 52.5 μM4,4′-dihydroxystilbene. Due to the higher cell toxicity of the stilbene,cells were not incubated in the presence of 75 μM dihydroxystilbene.

[0118] Upon addition of the medium to the cultures and at 24 hours timeperiods thereafter, i.e., 0 hours, 24 hours, 48 hours, and 72 hoursafter addition of the drug, cells and medium were frozen at −70° C.Samples were then thawed, sonicated and titrated on Vero cells todetermine the number of plaque forming units (pfu's) of virus producedby each culture.

[0119] As shown in FIG. 4, the number of pfu's produced in the controlcultures infected with an moi of 1 reaches peak production atapproximately 24 hours after infection. At this time, the system isexhausted, i.e., active virus has infected and destroyed not only thosecells infected during the initial one hour of incubation but also thosecells which became infected with virus released by theinitially-infected cells. The lack of increase observed in the controlcultures at 72 hours treatment indicates that the virus production haspeaked, due to the lack of viable cells in which to reproduce.

[0120] As shown in FIG. 4, treatment of cells with 75 μM4,4′-dihydroxytolan inhibited formation of infectious virus particles inHSV-1 infected cells by more than 99% at 24 hours. By 72 hours,infectious HSV particles were virtually undetectable in culturescontinuously incubated in the presence of 75 μM 4,4′-dihydroxytolan,Treatment with 50 μM 4,4′-dihydroxytolan reduced new virus production byonly 95%. In contrast, treatment of the HSV infected cells with 50 μM4,4′-dihydroxystilbene had no effect on virus production. (data notshown) These results also demonstrate that inhibition of virusreplication by the dihydroxylated tolan is dose dependent.

EXAMPLE 4 Inhibiting Formation of Infectious HSV-1 Particles byContacting Cells with a Hydroxylated Tolan Prior to or During an EarlyStage in Replication

[0121] Vero cell cultures were infected with HSV-1 as described above inExample 3 except that the cells were infected with virus at an moi of10. Under these conditions nearly all of the cells are infected withvirus during the initial one hour incubation period. Following removalof unattached virus, the virally-infected cultures were incubated incontrol medium lacking a hydroxylated tolan or medium to which 105 μM4,4′-dihydroxytolan had been added at 1, 3, 6, or 9 hours after removalof the unattached virus. At 24 hours after infection, the number ofpfu's present in the cells and medium of untreated and hydroxylatedtolan-treated cultures was determined.

[0122] The results presented in FIG. 5 demonstrate that the hydroxylatedtolan is most effective when administered to virally-infected cellsduring the early stages of viral replication. In cultures treated with105 μM 4,4′-dihydroxytolan at one hour after infection, production ofvirus was reduced by more than 99%. In cultures treated with 105 μM4,4′-dihydroxytolan at 3 or 6 hours after infection, the production ofvirus was inhibited by approximately 90%. When the hydroxylated tolanwas added 9 hours after infection, formation of infectious virusparticles was not inhibited.

[0123] To determine whether hydroxylated tolans block formation ofinfectious herpes virus particles by directly inactivating the virus, astandard inoculum of HSV-1 was mixed with 105 μM 4,4′-dihydroxytolan inmedium, with 0.2% DMSO in medium, or with media alone and placed at roomtemperature. The number of residual pfu's present at 1, 10, 30, and 60minutes after addition of each respective solution to the virus wasdetermined by plaque assay. The results demonstrated that thehydroxylated tolan did not directly inactivate HSV (data not shown).

[0124] Studies also demonstrated that the hydroxylated tolan did notprevent attachment of HSV-1 to cells. (data not shown)

EXAMPLE 5 Inhibiting HSV Replication

[0125] Vero cells were grown to confluence and infected with HSV-1 at anmoi of 1 and then incubated in media lacking a hydroxylated tolan(control cultures) or in media containing 105 μM 4,4′-dihydroxytolan.One set of infected cells was maintained in the dihydroxylatedtolan fora period of 72 hours. In another set of infected cells thedihydroxytolan-containing media was replaced with media lacking ahydroxylated tolan at 24 hours. In another set of cells thetolan-containing media was replaced with media lacking the hydroxylatedtolan at 48 hours after infection. The number of infectious HSVparticles produced by each set of infected cells was determined byplaque assay.

[0126] The results shown in FIG. 6 demonstrate that the inhibitoryeffect of 105 μM 4,4′-dihydroxytolan on HSV replication in virusinfected cells is reversible. Accordingly, continuous treatment ofHSV-infected cells with the hydroxylated tolan maintains the virus in anon-infectious state. Discontinuing treatment with the hydroxylatedtolan allows replication of the virus to proceed in what appears to be anormal fashion. The results presented in FIG. 6 also suggest that HSVreplication in the hydroxylated tolan treated cells was blocked at anearly phase, i.e., replication of HSV had not progressed past the stagewhere cells are so damaged that they are unable to support replicationof this herpes virus.

[0127] The results presented in FIG. 6 also indicate that exposure ofmammalian cells to 105 μM 4,4′-dihydroxytolan for a prolonged period oftime does not kill the cells. Cell viability studies confirmed thattreatment of uninfected Vero cells with 105 μM 4,4′-dihydroxytolan for24 hours was not toxic.

EXAMPLE 6 Characterizing Viral Proteins Produced in the Presence of aPolyhydroxylated Tolan

[0128] ICP-4 and ICP-27 are immediate-early regulatory proteins of HSV-1that are required for efficient replication of this virus. To determinewhether ICP-4 or ICP-27 production is altered by treatment with ahydroxylated tolan, separate cultures of Vero cells were infected withHSV-1 at an moi of 1 and incubated in control medium or mediumcontaining 105 μM 4,4′-dihydroxytolan for 24 hours. Infected cells werescraped from the flask, collected by centrifugation, and resuspended incold tris-buffered saline, pelleted by centrifugation, and the cellpellet frozen at −70° C. Proteins were extracted from the thawedpellets, separated by 6-15% SDS-PAGE, and assayed on a Western blot byreacting with mouse monoclonal antibody to ICP-4 or ICP-27 from GoodwinInstitute for Cancer Research Inc., FL.

[0129] As shown in FIGS. 7 and 8, treatment of HSV-infected cells withthe dihydroxytolan significantly reduced synthesis of ICP-4 and ICP-27,two major regulatory protein. These results confirm that treatment witha hydroxylated-tolan inhibits synthesis of herpes viruses at an earlyphase in the replicative scheme. These results also indicate thatcultured cells treated with a hydroxylated tolan are a useful modelsystem for characterizing the herpes virus gene products that are madeduring the immediate early phase and early phase of HSV replication.

EXAMPLE 8 Cell Toxicity of Tolans

[0130] Cell toxicity of the tolans 4,4′-dihydroxytolan (“Tolan-5”),3,4′,5-trihydroxytolan (“Tolan-10”) and4-hydroxy-4′-trifluoromethyltolan (“Tolan-11”) was determined using anMTT assay. In this assay, cells are exposed to MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, which istaken into the cells and reduced by mitochondrial dehydrogenase to apurple formazan, a large molecule which is unable to pass through intactcell membranes, and therefore accumulates in healthy cells. The abilityof cells to reduce MTT is an indication of mitochondrial integrity andactivity, which may be interpreted as a measure of viability.Solubilization of the cells results in the liberation of the productwhich can readily be detected spectrophotometrically.

[0131] Various concentrations of Tolan-5, Tolan-10 and Tolan-11 wereadded to 96 well tissue culture plates that had been seeded with Verocells. The plates were then incubated at 37° C. in 5% CO₂/95% air for24, 48 and 72 hours. At the end of each time point, 50 μl of MTT (1mg/ml) was added to the tissue culture plates which were then incubatedfor an additional four hours. At the end of that time, dimethylsulfoxide (DMSO) was added to dissolve the cells and formazan, and theplates were read spectrophotometrically at 540 nm. From this data a CD₅₀or the concentration of the respective tolan required to render 50% ofthe cells non-viable was calculated. Presented in Table I below are thetoxicity results for Tolan-5, Tolan-10, and Tolan-11 measured at 24, 48and 72 hours of treatment. TABLE 1 CD₅₀ for hydroxylated Tolans ofExamples 1 and 2: Toxicity was measured using a MTT colorimetric assayat 48 hours in Vero cells, and reported as the cytotoxic dose (CD₅₀ inμM, based on a 50% reduction in cell viability. CD₅₀ in μM Time in hoursName 24 48 72 Tolan-5 — 106 84.0 Tolan-10 >200 >200 >200 Tolan-11 72.148.2 47.1

[0132] As shown in Table 1, Tolan-10 showed very little toxicity. Theestimated CD₅₀ for this trihydoxytolan is >200 μm . Thehydroxytrfluoromethyltolan, Tolan-11, was more toxic than the otherhydroxylated tolans and is, thus, less preferred.

EXAMPLE 9 Inhibition of HSV-1 Replication In Vitro

[0133] Tissue culture cells were infected with HSV-1 and incubated insolutions containing Tolan-5, Tolan-10 and Tolan-11. The concentrationsused ranged from one-half to three-quarters of the calculated CD₅₀ forthe respective tolan at 48 hours of treatment.

[0134] Specifically, Vero cells were infected with HSV-1 at amultiplicity of infection of one for one hour. At the end of that time,the cultures were rinsed with media and fresh media containing variousconcentration of the respective tolan were added. Infected controlcultures were incubated in media lacking hydroxylated tolan. At 1, 24,48, and 72 hours after infection, samples were frozen at −70° C. untilassayed for new virus production. Virus production was quantified by theplaque assay for each time point of infection. The results, which areexpressed as plaque forming units per milliliter (pfu/ml).

[0135] As shown in FIGS. 9A-C, Tolan-5, Tolan-10, and Tolan-11 allinhibited HSV-1 production, but to varying degrees. Tolan-5 (FIG. 9A)was more inhibitory to the virus at 75 μm that at 50 μm. Tolan-10 (FIG.9B) was equally effective against the virus at all concentrations, whichranged from a high of 350 μm to a low of 175 μm. Tolan-11 had little tono effect at concentration of less than 48 μm.

[0136] When inhibition occurred, it was seen with all tolans at theearliest time point assayed, which was 24 hours after cell infection.Once suppressed, the virus did not recover over the 72 hour time periodthe cells were tested for virus production.

EXAMPLE 10 Inhibition of HSV In Vivo

[0137] Female SKH-1 hairless mice 4-5 weeks old were lightlyanesthetized and a scratch approximately 2-3 mm long and 0.25 mm deepwas made on the lateral dorsal aspect of the neck. The scratch wasinfected with 10⁶ plaque forming units of HSV-1. One hour later, theinfected scratch area was treated with a 5% or 10% solution of Tolan-10in DMSO. This treatment was repeated three times a day for five days.There were at least six mice in each treatment group. Untreated mice andmice treated with DMSO only were used as controls.

[0138] Beginning on day 1 and continuing for 10-11 days, the scratchsite was examined daily for evidence of an HSV infection and scoredaccording to the following schedule:

[0139] 0=no visible change on skin by scratch

[0140] 1+=papules around scratch

[0141] 2+=papules around scratch ulcerate with scab formation

[0142] 3+=ulcerative zosterform spread along peripheral nerves to flankof animal

[0143] 4+=ulcerative lesions open on flank

[0144] 5+=death/sacrifice

[0145] The data was grouped according to treatment and statisticallyanalyzed for significance.

[0146] As shown in FIGS. 10 and 11, Tolan-10 at concentrations of 5 and10% significantly reduced the severity of HSV lesions when compared tountreated controls or animals that were treated with DMSO only. Thisdifference was evident as early as three days after infection. Althoughnot significantly different, 10% Tolan-10 appeared to be slightly moreeffective than 5% Tolan-10, particularly on days 6-10. At day 11,however, there was no difference between the two treatment groups.

[0147] Survival data is shown in FIG. 11. While 50% of the animals inthe DMSO control and no treatment control groups died within 11 daysafter infection, none of the animals treated with 5% or 10% tolanperished during this time period.

EXAMPLE 11 Inhibiting Growth of Neisseria gonorrhea by Treatment withTolan-10

[0148] Various concentrations of the trihydroxytolan Tolan-10 wereincorporated into chocolate agar plates that were prepared usingstandard procedures. The trihydroxylated tolan was first dissolved inDMSO and medium and then added to liquid chocolate agar, which was thenpoured into petri plates and allowed to solidify. The highestconcentration of DMSO in the agar was 0.5%. Control plates containingchocolate agar and DMSO at a final concentration of 0.5% were alsoprepared.

[0149]Neisseria gonorrhea CDC 98 was obtained from Difco Laboratories.The authenticity of the bacterium was confirmed utilizing standardmicrobiological techniques of identification. Cultures of the bacterialisolate were inoculated onto fresh plates of solidified chocolate agarand then 24 hours later, a suspension was made from isolated colonies.

[0150] 10 μl aliquots of the suspension were spread evenly across thesurface of solidified control chocolate agar lacking tolan and thesurface of solidified chocolate agar containing Tolan-10 at finalconcentrations ranging from 1 to 125 μg/ml. Thereafter, the agar plateswere incubated at 37° C. with or without 5% CO₂. All plates werevisually examined for growth of the bacterium 24 hours later todetermine the concentration of the tri-hydroxylated tolan that inhibitsgrowth by 50% (MIC₅₀) as well as the concentration which inhibits anyvisible growth (MIC₁₀₀).

[0151] The effect of the same concentrations of Tolan-10 on the growthof Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes,Pseudomonas aeruginosa, Neisseria meningiditis and Candida albicans wasalso determined. As shown in Table II below, Tolan 10 selectivelyinhibited Neisseria gonorrhoeae. The IC₅₀ value of the tri-hydroxylatedtolan for this bacterium at 24 hours of treatment was 50 μg/ml and theIC₁₀₀ value was 100 μg/ml. In contrast, Tolan 10, at the highestconcentration tested, had no effect on the other microorganisms listed.TABLE 2 IC's of Tolan-10 against bacteria and C. albicans MicroorganismIC₅₀ (mg/L) IC₁₀₀ (mg/L) N. gonorrhoeae 50 100 N. meningitides >125 >125E. coli >125 >125 S. aureus >125 >125 S. pyogenes >125 >125 P.aeruginosa >125 >125 C. albicans >125 >125

What is claimed is:
 1. A method of inhibiting formation of infectiousherpes virus particles in a host cell comprising: administering ahydroxylated tolan to the host cell.
 2. The method of claim 1 whereinthe hydroxylated tolan is administered in an amount effective to blockreplication of the herpes virus in an infected cell.
 3. The method ofclaim 1 wherein the hydroxylated tolan is administered prior to orwithin 6 hours of infection of the host cell with the herpes virus. 4.The method of claim 1 wherein the hydroxylated tolan is apolyhydroxylated tolan and the herpes virus is selected from the groupconsisting of HSV-1, HSV-2, CMV and VZV.
 5. The method of claim 1wherein the hydroxylated tolan is a dihydroxylated, or trihydroxylatedtolan and the herpes virus is selected from the group consisting ofHSV-1 and HSV-2.
 6. A method of treating a subject having or suspectedof having an infection induced by a herpes virus selected from the groupconsisting of HSV-1, HSV-2, CMV and VZV, said method comprisingadministering a pharmaceutical composition comprising a therapeuticallyeffective amount of a hydroxylated tolan to the subject.
 7. The methodof claim 6 wherein the hydroxylated tolan is a polyhydroxylated tolan.8. The method of claim 6 wherein the hydroxylated tolan is adihydroxylated or trihydroxylated tolan.
 9. The method of claim 6wherein the pharmaceutical composition further comprises a topicalcarrier and administration is by topical administration to the skin. 10.The method of claim 6 wherein administration is to the eye.
 11. Themethod of claim 6 wherein administration is to the oral cavity or lips.12. The method of claim 6 wherein administration is by vaginal insertionor anal insertion.
 13. The method of claim 9 wherein the herpes virusinfection is caused by HSV-1 or HSV-2.
 14. The method of claim 6 whereinthe pharmaceutical composition is administered to an infected siteduring the prodromal stage of infection.
 15. The method of claim 6wherein the pharmaceutical is applied at or proximate a known site ofinfection or a site which is suspected of being infected.
 16. A topicalcomposition for reducing the symptoms of a herpes virus infection, saidherpes virus being selected from the group consisting HSV-1, HSV-2, VZVand CMV, said topical composition comprising: (a) a therapeuticallyeffective amount of a hydroxylated tolan; and (b) a pharmaceuticallyacceptable carrier.
 17. The topical composition of claim 16 wherein saidhydroxylated tolan is a polyhydroxylated tolan.
 18. The topicalcomposition of claim 16 wherein said hydoxylated tolan is adihydroxylated or trihydroxylated tolan.
 19. The topical composition ofclaim 16 wherein said topical composition is formulated foradministration to the skin, reproductive tract, oral cavity or eye of asubject in need of the same.
 20. A method of inhibiting growth of aNeisseria gonorrhea, said method comprising contacting the Neisseriagonorrhea with a hydroxylated tolan.
 21. The method of claim 20 whereinthe hydroxylated tolan is a dihydroxylated or trihydroxylated tolan. 22.The method of claim 20 wherein the hydroxylated tolan is in apharmaceutical composition which is administered to a human subject. 23.The method of claim 22 wherein the pharmaceutical composition isadministered to the eye, oropharynx, genitalia, or rectum of thesubject.